Project Summary The long-term goal of the project is to develop a general strategy for improving clinical applications of electrotransfection (ET). The technology has been widely used for gene delivery in different applications, such as genome and epigenome editing, cell and gene therapies, and vaccination for prevention of diseases. However, the technique is currently limited by its low efficiency. Only a tiny fraction of plasmid DNA (pDNA) molecules in extracellular space can be delivered into the nucleus of cells for target gene expression. As a result, ET requires to use buffers with high pDNA concentration and electric pulses with high energy, which can cause cytotoxicity and induce undesired immune responses in cells. To improve the efficiency, the overall objective of the proposed study is to understand molecular mechanisms of pDNA transport in cells. Understanding the mechanisms is critical for development of a general strategy for improving the efficiency of ET, in which intracellular pathways will be manipulated to enhance pDNA transport to the nucleus, and reduce its degradation in the cytoplasm. The central hypothesis in the study is that intracellular transport of electrotransfected pDNA is mediated by vesicles in noncanonical pathways that overlap with those for endocytosis and autophagy. To test the hypothesis, the study will investigate specific pathways involved in cellular uptake and intracellular transport of electrotransfected pDNA (Aim 1). The investigation will be based on quantitative analysis of spatial and temporal distributions of pDNA in cells and its associations with endocytic and autophagic markers. Meanwhile, components in intracellular pathways will be manipulated to identify those that can be used to enhance ET efficiency and cell viability (Aim 2). The manipulation will include treatment of cells with different electric pulses and pharmacological agents, or changing expression levels of specific genes in cells prior to or post ET. The investigations in Aims 1 and 2 will use cells from two dimensional (2D) culture. To understand how ET in 2D differ from that in 3D microenvironment, the proposed study will investigate mechanisms of ET in 3D cell constructs (Aim 3). Results from the mechanistic study will be used, as a proof of principle, to enhance electrogene transfer in solid tumors in vivo (Aim 3). Taken together, the proposed study will reveal new mechanisms of ET in both 2D and 3D models, and develop a more general strategy for improving ET efficiency and cell viability for all cell types. The increase in efficiency will also decrease the amount of pDNA required for ET, thereby reducing undesired innate immune responses to ET. Compared to empirical, trial-and-error approaches used currently in the literature, the new strategy will be more efficient, versatile, and practical, which is critical for improving ET in clinical applications.